Bioengineered vascular graft support prostheses

ABSTRACT

The invention is directed to bioengineered vascular graft support prostheses prepared from cleaned tissue material derived from animal sources. The bioengineered graft prostheses of the invention are prepared using methods that preserve cell compatibility, strength, and bioremodelability of the processed tissue matrix. The bioengineered graft prostheses are used for implantation, repair, or for use in a mammalian host.

FIELD OF THE INVENTION

[0001] This invention is in the field of tissue engineering. Theinvention is directed to bioengineered graft prostheses prepared fromcleaned tissue material derived from animal sources. The bioengineeredgraft prostheses of the invention are prepared using methods thatpreserve cell compatibility, strength, and bioremodelability of theprocessed tissue matrix. The bioengineered graft prostheses are used forimplantation, repair, or for use in a mammalian host.

BRIEF DESCRIPTION OF THE BACKGROUND OF THE INVENTION

[0002] The field of tissue engineering combines the methods ofengineering with the principles of life science to understand thestructural and functional relationships in normal and pathologicalmammalian tissues. The goal of tissue engineering is the development andultimate application of biological substitutes to restore, maintain, andimprove tissue functions.

[0003] Collagen is the principal structural protein in the body andconstitutes approximately one-third of the total body protein. Itcomprises most of the organic matter of the skin, tendons, bones, andteeth and occurs as fibrous inclusions in most other body structures.Some of the properties of collagen are its high tensile strength; itslow antigenicity, due in part to masking of potential antigenicdeterminants by the helical structure; and its low extensibility,semipermeability, and solubility. Furthermore, collagen is a naturalsubstance for cell adhesion. These properties and others make collagen asuitable material for tissue engineering and manufacture of implantablebiological substitutes and bioremodelable prostheses.

[0004] Methods for obtaining collagenous tissue and tissue structuresfrom explanted mammalian tissues and processes for constructingprosthesis from the tissue, have been widely investigated for surgicalrepair or for tissue or organ replacement. It is a continuing goal ofresearchers to develop prostheses that can successfully be used toreplace or repair mammalian tissue.

SUMMARY OF THE INVENTION

[0005] Biologically-derived collagenous materials such as the intestinalsubmucosa have been proposed by a many of investigators for use intissue repair or replacement. Methods for mechanical and chemicalprocessing of the proximal porcine jejunum to generate a single,acellular layer of intestinal collagen (ICL) that can be used to formlaminates for bioprosthetic applications are disclosed. The processingremoves cells and cellular debris while maintaining the native collagenstructure. The resulting sheet of processed tissue matrix is used tomanufacture multi-layered laminated constructs with desiredspecifications. We have investigated the efficacy of laminated patchesfor soft tissue repair as well as the use of entubated ICL as a supportfor vascular grafts. This material provides the necessary physicalsupport and is able to integrate into the surrounding native tissue andbecome infiltrated with host cells. In vivo remodeling does notcompromise mechanical integrity. Intrinsic and functional properties ofthe implant, such as the modulus of elasticity, suture retention and UTSare important parameters which can be manipulated for specificrequirements by varying the number of ICL layers and the crosslinkingconditions.

DETAILED DESCRIPTION OF THE INVENTION

[0006] This invention is directed to a tissue engineered prostheses,which, when implanted into a mammalian host, can serve as a functioningrepair, augmentation, or replacement body part or tissue structure, andwill undergo controlled biodegradation occurring concomitantly withremodeling by the host's cells. The prosthesis of this invention, whenused as a replacement tissue, thus has dual properties: First, itfunctions as a substitute body part, and second, while still functioningas a substitute body part, it functions as a remodeling template for theingrowth of host cells. In order to do this, the prosthetic material ofthis invention is a processed tissue matrix developed from mammalianderived collagenous tissue that is able to be bonded to itself oranother processed tissue matrix to form a prosthesis for grafting to apatient.

[0007] The invention is directed toward methods for making tissueengineered prostheses from cleaned tissue material where the methods donot require adhesives, sutures, or staples to bond the layers togetherwhile maintaining the bioremodelability of the prostheses. The terms,“processed tissue matrix” and “processed tissue material”, mean native,normally cellular tissue that has been procured from an animal source,preferably a mammal, and mechanically cleaned of attendant tissues andchemically cleaned of cells, cellular debris, and rendered substantiallyfree of non-collagenous extracellular matrix components. The processedtissue matrix, while substantially free of non-collagenous components,maintains much of its native matrix structure, strength, and shape.Preferred compositions for preparing the bioengineered grafts of theinvention are animal tissues comprising collagen, including, but notlimited to: intestine, fascia lata, pericardium, dura mater, and otherflat or planar structured tissues that comprise a collagenous tissuematrix. The planar structure of these tissue matrices makes them able tobe easily cleaned, manipulated, and assembled in a way to prepare thebioengineered grafts of the invention. Other suitable collagenous tissuesources with the same flat sheet structure and matrix composition may beidentified by the skilled artisan in other animal sources.

[0008] A more preferred composition for preparing the bioengineeredgrafts of the invention is an intestinal collagen layer derived from thetunica submucosa of small intestine. Suitable sources for smallintestine are mammalian organisms such as human, cow, pig, sheep, dog,goat, or horse while small intestine of pig is the preferred source.

[0009] The most preferred composition for preparing the prosthesis ofthe invention is a processed intestinal collagen layer derived thetunica submucosa of porcine small intestine. To obtain the processedintestinal collagen layer, the small intestine of a pig is harvested andattendant mesenteric tissues are grossly dissected from the intestine.The tunica submucosa is preferably separated, or delaminated, from theother layers of the small intestine by mechanically squeezing the rawintestinal material between opposing rollers to remove the muscularlayers (tunica muscularis) and the mucosa (tunica mucosa). The tunicasubmucosa of the small intestine is harder and stiffer than thesurrounding tissue, and the rollers squeeze the softer components fromthe submucosa. In the examples that follow, the tunica submucosa wasmechanically harvested from porcine small intestine using a Bitterlinggut cleaning machine and then chemically cleaned to yield a cleanedtissue matrix. This mechanically and chemically cleaned intestinalcollagen layer is herein referred to as “ICL”.

[0010] The processed ICL is essentially acellular telopeptide collagen,about 93% by weight dry, with less than about 5% dry weightglycoproteins, glycosaminoglycans, proteoglycans, lipids,non-collagenous proteins and nucleic acids such as DNA and RNA and issubstantially free of cells and cellular debris. The processed ICLretains much of its matrix structure and its strength. Importantly, thebioremodelability of the tissue matrix is preserved in part by thecleaning process as it is free of bound detergent residues that wouldadversely affect the bioremodelability of the collagen. Additionally,the collagen molecules have retained their telopeptide regions as thetissue has not undergone treatment with enzymes during the cleaningprocess.

[0011] The collagen layers of the prosthetic device may be from the samecollagen material, such as two or more layers of ICL, or from differentcollagen materials, such as one or more layers of ICL and one or morelayers of fascia lata.

[0012] The processed tissue matrices may be treated or modified, eitherphysically or chemically, prior to fabrication of a bioengineered graftprosthesis. Physical modifications such as shaping, conditioning bystretching and relaxing, or perforating the cleaned tissue matrices maybe performed as well as chemical modifications such as binding growthfactors, selected extracellular matrix components, genetic material andother agents that would affect bioremodeling and repair of the body partbeing treated, repaired, or replaced.

[0013] As ICL is the most preferred starting material for the productionof the bioengineered graft prostheses of the invention, the methodsdescribed below are the preferred methods for producing bioengineeredgraft prostheses comprising ICL.

[0014] In the most preferred embodiment, the tunica submucosa of porcinesmall intestine is used as a starting material for the bioengineeredgraft prosthesis of the invention. The small intestine of a pig isharvested, its attendant tissues removed and then mechanically cleanedusing a gut cleaning machine which forcibly removes the fat, muscle andmucosal layers from the tunica submucosa using a combination ofmechanical action and washing using water. The mechanical action can bedescribed as a series of rollers that compress and strip away thesuccessive layers from the tunica submucosa when the intact intestine isrun between them. The tunica submucosa of the small intestine iscomparatively harder and stiffer than the surrounding tissue, and therollers squeeze the softer components from the submucosa. The result ofthe machine cleaning was such that the submucosal layer of the intestinesolely remained.

[0015] After mechanical cleaning, a chemical cleaning treatment isemployed to remove cell and matrix components, preferably performedunder aseptic conditions at room temperature. The intestine is then cutlengthwise down the lumen and then cut into approximately 15 cm squaresheet sections. Material is weighed and placed into containers at aratio of about 100:1 v/v of solution to intestinal material. In the mostpreferred chemical cleaning treatment, such as the method disclosed inInternational PCT Application WO 98/49969, the disclosure of which isincorporated herein, the collagenous tissue is contacted with achelating agent, such as ethylenediaminetetraacetic tetrasodium salt(EDTA) under alkaline conditions, preferably by addition of sodiumhydroxide (NaOH); followed by contact with an acid where the acidcontains a salt, preferably hydrochloric acid (HCl) containing sodiumchloride (NaCl); followed by contact with a buffered salt solution suchas 1 M sodium chloride (NaCl)/10 mM phosphate buffered saline (PBS):finally followed by a rinse step using water.

[0016] Each treatment step is preferably carried out using a rotating orshaking platform. After rinsing, the water is then removed from eachcontainer and the ICL is blotted of excess water using sterile absorbenttowelettes. At this point, the ICL may be stored frozen at −80° C., at 4° C. in sterile phosphate buffer, or dry until use in fabrication of aprosthesis. If to be stored dry, the ICL sheets are flattened on asurface such as a flat plate, preferably a plate or membrane, such as arigid polycarbonate sheet, and any lymphatic tags from the abluminalside of the material are removed using a scalpel, and the ICL sheets areallowed to dry in a laminar flow hood at ambient room temperature andhumidity.

[0017] The ICL is a planar sheet structure that can be used to fabricatevarious types of constructs to be used as a prosthesis with the shape ofthe prosthesis ultimately depending on its intended use. To formprostheses of the invention, the constructs must be fabricated using amethod that preserves the bioremodelability of the processed matrixmaterial but also is able to maintain its strength and structuralcharacteristics in its performance as a replacement tissue. Theprocessed tissue matrix sheets are layered to contact another sheet ortubulated and wrapped over on itself. The area of contact is a bondingregion where layers contact. The bonding region must be able towithstand suturing and stretching during implantation and in the initialhealing phase until the patients cells populate and subsequentlybioremodel the prosthesis to form a new tissue. When used as a conduitor a duct, the bonding region must be able to withstand pressures of thematter it contains or is passing, particularly when used as a vasculargraft under the systolic and diastolic pressures of systemic blood flow.

[0018] In a preferred embodiment, the prosthetic device of thisinvention is a tubular construct formed from a single, generallyrectangular sheet of processed tissue matrix. The processed tissuematrix is rolled so that one edge meets and overlaps an opposing edge.The overlap serves as a bonding region. As used herein, “bonding region”means an area of contact between tow or more layers of the same ordifference processed tissue matrix treated in a manner such that thelayers are superimposed on each other and are sufficiently held togetherby self-lamination and chemical linking. For instance, a multilayersheet construct of ICL is used to repair body wall structures such as apericardial patch or a hernia repair device, tubular constructs can beused to repair tubular organs that serve as conduits such as vasculatureor digestive tract structures or used as a neuron growth tube to guidenerve regeneration. They may also be implanted for tissue bulking andaugmentation. A number of layers of ICL may be incorporated in theconstruct for bulking or strength indications. Prior implantation, thelayers may be further treated or coated with collagen or otherextracellular matrix components, hyaluronic acid, or heparin, growthfactors, peptides or cultured cells.

[0019] In a preferred embodiment, an ICL sheet is formed into a tubularprosthesis. The ICL tube may be fabricated in various diameters,lengths, and number of layers and may incorporate other componentsdepending on the indication for its use. The tubular ICL construct maybe used as a vascular graft. For this indication, the graft comprises atleast one layer with at least a 5% overlap to act as a bonding regionthat forms a tight seam and the luminal surface is preferably treatedwith heparin or an agent that prevents thrombosis. Other means forpreventing thrombosis are known in the art of fabricating vascularconstructs. In another vascular indication, the tubular ICL construct isformed on a metal stent to provide a cover for the stent. Whenimplanted, the ICL benefits the recipient by providing a smoothprotective covering for the stent, to prevent additional damage to hosttissue during deployment. Tubular ICL prostheses may also be used torepair or replace other normally tubular structures such asgastrointestinal tract sections, urethra, ducts, etc. It may also beused in nervous system repair when fabricated into a nerve growth tubepacked with extracellular matrix components, growth factors, or culturedcells.

[0020] In another preferred vascular indication, the tubular ICLconstruct may be used as an external stent in cases where damaged ordiseased blood vessels or autograft vessels require exterior support. Inone such indication, vein autografts are transplanted within the bodyand external support for the transplanted vein is desired. Before thetransplanted vessel is fully anastomosed to the existing vasculature,the vessel is first passed through the lumen of an ICL tube. The vesselis then anastomosed and then the ends of the ICL tube are then securedto maintain the position of the construct.

[0021] To form a tubular construct, a mandrel is chosen with a diametermeasurement that will determine the diameter of the formed construct.The mandrel is preferably cylindrical or oval in cross section and madeof glass, stainless steel or of a nonreactive, medical gradecomposition. The mandrel may be straight, curved, angled, it may havebranches or bifurcations, or a number of these qualities. The number oflayers intended for the tubular construct to be formed corresponds withthe number of times an ICL is wrapped around a mandrel and over itself.The number of times the ICL can be wrapped depends on the width of theprocessed ICL sheet. For a two layer tubular construct, the width of thesheet must be sufficient for wrapping the sheet around the mandrel atleast twice. It is preferable that the width be sufficient to wrap thesheet around the mandrel the required number of times and an additionalpercentage more as an overlap to serve as a bonding region, for a singlelayer construct, preferably between about 5% to about 20% of the mandrelcircumference to serve as a bonding region and to form a tight seam.Similarly, the length of the mandrel will dictate the length of the tubethat can be formed on it. For ease in handling the construct on themandrel, the mandrel should be longer than the length of the constructso the mandrel, and not the construct being formed, is contacted whenhandled.

[0022] The ICL has a sidedness quality derived from its native tubularstate. The ICL has two opposing surfaces: a mucosal surface that facedthe intestinal lumen and a serosal surface that previously had exteriorintestinal tissues attached to it, such as mesentery and vasculature. Ithas been found that these surfaces have characteristics that can affectpost-operative performance of the prosthesis but can be leveraged forenhanced device performance. In the formation of a tubular construct foruse in as a vascular graft, it is preferred that the mucosal surface ofthe material be the luminal surface of the tubular graft when formed. Invascular applications, having the mucosal surface contact the blood flowprovides an advantage as it has some nonthrombogenic properties that arepreferred to prevent occlusion of the graft when it has been implantedin a patient. In other tubular constructs, the orientation of the layerof the construct depends on the intended use.

[0023] It is preferred that the mandrel is provided with a covering of anonreactive, medical grade quality, elastic, rubber or latex material inthe form of a sleeve. While a tubular ICL construct may be formeddirectly on the mandrel surface, the sleeve facilitates the removal ofthe formed tube from the mandrel and does not adhere to, react with, orleave residues on the ICL. To remove the formed construct, the sleevemay be pulled from one end off the mandrel to carry the construct fromthe mandrel with it. Because the processed ICL only lightly adheres tothe sleeve and is more adherent to other ICL layers, fabricating ICLtubes is facilitated as the tubulated contract may be removed from themandrel without stretching or otherwise stressing or risking damage tothe construct. In the most preferred embodiment, the sleeve comprisesKRATON® (Shell Chemical Company), a thermoplastic rubber composed ofstyrene-ethylene/butylene-styrene copolymers with a very stablesaturated midblock.

[0024] For simplicity in illustration, a two-layer tubular constructwith a 4 mm diameter and a 10% overlap is formed on a mandrel havingabout a 4 mm diameter. The mandrel is provided with a KRATON® sleeveapproximately as long as the length of the mandrel and longer than theconstruct to be formed on it. A sheet of ICL is trimmed so that thewidth dimension is about 28 mm and the length dimension may varydepending on the desired length of the construct. In the sterile fieldof a laminar flow cabinet, the ICL is then formed into an ICL collagentube by the following process. The ICL is moistened along one edge andis aligned with the sleeve-covered mandrel and, leveraging the adhesivenature of the ICL, it is “flagged” along the length of thesleeve-covered mandrel and dried in position for at least 10 minutes ormore. The flagged ICL is then hydrated and wrapped around the mandreland then over itself one full revolution plus 10% of the circumference,for a 110% overlap, to serve as a bonding region and to provide a tightseam. To obtain a tubular construct with the mucosal side of the ICL asthe lumen of the formed construct, the mucosal side of the ICL ismoistened

[0025] The constructs are then crosslinked together by contacting themwith a crosslinking agent, preferably a chemical crosslinking agent thatpreserves the bioremodelability of the ICL material. As mentioned above,the dehydration brings the extracellular matrix components of adjacentICL layers together for crosslinking those layers of the wrap togetherto form chemical bonds between the components and thus bond the layerstogether. Alternatively, the constructs may be rehydrated beforecrosslinking by contacting an aqueous solution, preferably water, bytransferring them to a room temperature container containing rehydrationagent for at least about 10 to about 15 minutes to rehydrate the layerswithout separating or delaminating them. Crosslinking the bondedprosthetic device also provides strength and durability to the device toimprove handling properties. Various types of crosslinking agents areknown in the art and can be used such as ribose and other sugars,oxidative agents and dehydrothermal (DHT) methods. A preferredcrosslinking agent is 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC). In an another preferred method,sulfo-N-hydroxysuccinimide is added to the EDC crosslinking agent asdescribed by Staros, J. V., Biochem. 21, 3950-3955, 1982. Besideschemical crosslinking agents, the layers may be bonded together by othermeans such as with fibrin-based glues or medical grade adhesives such aspolyurethane, vinyl acetate or polyepoxy. In the most preferred method,EDC is solubilized in water at a concentration preferably between about0.1 mM to about 100 mM, more preferably between about 1.0 mM to about 10mM, most preferably at about 1.0 mM. Besides water, phosphate bufferedsaline or (2-[N-morpholino]ethanesulfonic acid) (MES) buffer may be usedto dissolve the EDC. In addition, other agents may be added to thesolution such as acetone or an alcohol may be added up to 99% v/v inwater to make crosslinking more uniform and efficient. EDC crosslinkingsolution is prepared immediately before use as EDC will lose itsactivity over time. To contact the crosslinking agent to the ICL, thehydrated, bonded ICL constructs are transferred to a container such as ashallow pan and the crosslinking agent gently decanted to the panensuring that the ICL layers are both covered and free-floating and thatno air bubbles are present under or within the layers of ICL constructs.The pan is covered and the layers of ICL are allowed to crosslink forbetween about 4 to about 24±2 hours after which time the crosslinkingsolution is decanted and disposed of.

[0026] Constructs are rinsed in the pan by contacting them with a rinseagent to remove residual crosslinking agent. A preferred rinse agent iswater or other aqueous solution. Preferably, sufficient rinsing isachieved by contacting the chemically bonded constructs three times withequal volumes of sterile water for about five minutes for each rinse. Ifthe constructs have not been removed from the mandrels, they may beremoved at this point by pulling the sleeves from the mandrels. Theconstructs are then allowed to dry and when dry, the sleeve may beremoved from the lumen of the constructs simply by pulling it out by oneof the free ends.

[0027] In embodiments where the construct will be used as a vasculargraft, the construct is rendered non-thrombogenic by applying heparin tothe lumen of the formed tube. Heparin can be applied to the prosthesis,by a variety of well-known techniques. For illustration, heparin can beapplied to the prosthesis in the following three ways. First,benzalkonium heparin (BA-Hep) isopropyl alcohol solution is applied tothe prosthesis by vertically filling the lumen or dipping the prosthesisin the solution and then air-drying it. This procedure treats thecollagen with an ionically bound BA-Hep complex. Second, EDC can be usedto activate the heparin and then to covalently bond the heparin to thecollagen fiber. Third, EDC can be used to activate the collagen, thencovalently bond protamine to the collagen and then ionically bondheparin to the protamine. Many other coating, bonding, and attachmentprocedures are well known in the art which could also be used.

[0028] Constructs are then terminally sterilized using means known inthe art of medical device sterilization. A preferred method forsterilization is by contacting the constructs with sterile 0.1%peracetic acid (PA) treatment neutralized with a sufficient amount of 10N sodium hydroxide (NaOH), according to U.S. Pat. No. 5,460,962, thedisclosure of which is incorporated herein. Decontamination is performedin a container on a shaker platform, such as 1 L Nalge containers, forabout 18±2 hours. Constructs are then rinsed by contacting them withthree volumes of sterile water for 10 minutes each rinse.

[0029] The constructs of the invention may also be sterilized usinggamma irradiation. Constructs are packaged in containers made frommaterial suitable for gamma irradiation and sealed using a vacuumsealer, which were in turn placed in hermetic bags for gamma irradiationbetween 25.0 and 35.0 kGy. Gamma irradiation significantly, but notdetrimentally, decreases Young's modulus and shrink temperature. Themechanical properties after gamma irradiation are still sufficient foruse in a range of applications and gamma is a preferred means forsterilizing as it is widely used in the field of implantable medicaldevices.

[0030] Tubular prostheses may be used, for example, to replace crosssections of tubular organs such as vasculature, esophagus, trachea,intestine, and fallopian tubes. These organs have a basic tubular shapewith an outer surface and an inner luminal surface. Flat sheets may alsobe used for organ support, for example, to support prolapsed orhypermobile organs by using the sheet as a sling for the organs, such asbladder or uterus. In addition, flat sheets and tubular structures canbe formed together to form a complex structure to replace or augmentcardiac or venous valves.

[0031] The bioengineered graft prostheses of the invention may be usedto repair or replace body structures that have been damaged or diseasedin host tissue. While functioning as a substitute body part or support,the prosthesis also functions as a bioremodelable matrix scaffold forthe ingrowth of host cells. “Bioremodeling” is used herein to mean theproduction of structural collagen, vascularization, and cellrepopulation by the ingrowth of host cells at a rate about equal to therate of biodegradation, reforming and replacement of the matrixcomponents of the implanted prosthesis by host cells and enzymes. Thegraft prosthesis retains its structural characteristics while it isremodeled by the host into all, or substantially all, host tissue, andas such, is functional as an analog of the tissue it repairs orreplaces.

[0032] The shrink temperature (° C.) of the tissue matrix prosthesis isan indicator of the extent of matrix crosslinking. The higher the shrinktemperature, the more crosslinked the material. Non-crosslinked ICL hasa shrink temperature of about 68±0.3° C. In the preferred embodiment,EDC crosslinked prostheses should have a shrink temperature betweenabout 68±0.3° C. to about 75±1° C.

[0033] The mechanical properties include mechanical integrity such thatthe prosthesis resists creep during bioremodeling, and additionally ispliable and suturable. The term “pliable” means good handling propertiesfor ease in use in the clinic.

[0034] The term “suturable” means that the mechanical properties of thelayer include suture retention which permits needles and suturematerials to pass through the prosthesis material at the time ofsuturing of the prosthesis to sections of native tissue, a process knownas anastomosis. During suturing, such prostheses must not tear as aresult of the tensile forces applied to them by the suture, nor shouldthey tear when the suture is knotted. Suturability of prostheses, i.e.,the ability of prostheses to resist tearing while being sutured, isrelated to the intrinsic mechanical strength of the prosthesis material,the thickness of the graft, the tension applied to the suture, and therate at which the knot is pulled closed. Suture retention for a highlycrosslinked flat 6 layer prosthesis crosslinked in 100 mM EDC and 50%acetone is at least about 6.5 N. Suture retention for a 2-layer tubularprosthesis crosslinked in 1 mM EDC in water is about 3.9 N±0.9 N. Thepreferred lower suture retention strength is about 2 N for a crosslinkedflat 2 layer prosthesis; a surgeon's pull strength when suturing isabout 1.8 N.

[0035] As used herein, the term “non-creeping” means that thebiomechanical properties of the prosthesis impart durability so that theprosthesis is not stretched, distended, or expanded beyond normal limitsafter implantation. As is described below, total stretch of theimplanted prosthesis of this invention is within acceptable limits. Theprosthesis of this invention acquires a resistance to stretching as afunction of post-implantation cellular bioremodeling by replacement ofstructural collagen by host cells at a faster rate than the loss ofmechanical strength of the implanted materials due from biodegradationand remodeling.

[0036] The processed tissue material of the present invention is“semi-permeable,” even though it has been layered and bonded.Semi-permeability permits the ingrowth of host cells for remodeling orfor deposition of agents and components that would affectbioremodelability, cell ingrowth, adhesion prevention or promotion, orblood flow. The “non-porous” quality of the prosthesis prevents thepassage of fluids intended to be retained by the implantation of theprosthesis. Conversely, pores may be formed in the prosthesis if aporous or perforated quality is required for an application of theprosthesis.

[0037] The mechanical integrity of the prosthesis of this invention isalso in its ability to be draped or folded, as well as the ability tocut or trim the prosthesis obtaining a clean edge without delaminatingor fraying the edges of the construct.

[0038] The following examples are provided to better explain thepractice of the present invention and should not be interpreted in anyway to limit the scope of the present invention. It will be appreciatedthat the device design in its composition, shape, and thickness is to beselected depending on the ultimate indication for the construct. Thoseskilled in the art will recognize that various modifications can be madeto the methods described herein while not departing from the spirit andscope of the present invention.

EXAMPLES Example 1

[0039] Chemical Cleaning of Mechanically Cleaned Porcine Small Intestine

[0040] The small intestine of a pig was harvested and mechanicallystripped, using a Bitterling gut cleaning machine (Nottingham, UK) whichforcibly removes the fat, muscle and mucosal layers from the tunicasubmucosa using a combination of mechanical action and washing usingwater. The mechanical action can be described as a series of rollersthat compress and strip away the successive layers from the tunicasubmucosa when the intact intestine is run between them. The tunicasubmucosa of the small intestine is comparatively harder and stifferthan the surrounding tissue, and the rollers squeeze the softercomponents from the submucosa. The result of the machine cleaning wassuch that the submucosal layer of the intestine solely remained. Theremainder of the procedure was performed under aseptic conditions and atroom temperature. The chemical solutions were all used at roomtemperature. The intestine was then cut lengthwise down the lumen andthen cut into 15 cm sections. Material was weighed and placed intocontainers at a ratio of about 100:1 v/v of solution to intestinalmaterial.

[0041] A. To each container containing intestine was added approximately1 L solution of 0.22 μm (micron) filter sterilized 100 mMethylenediaminetetraacetic tetrasodium salt (EDTA)/10 mM sodiumhydroxide (NaOH) solution. Containers were then placed on a shaker tablefor about 18 hours at about 200 rpm. After shaking, the EDTA/NaOHsolution was removed from each bottle.

[0042] B. To each container was then added approximately 1 L solution of0.22 μm filter sterilized 1 M hydrochloric acid (HCl)/1 M sodiumchloride (NaCl) solution. Containers were then placed on a shaker tablefor between about 6 to 8 hours at about 200 rpm. After shaking, theHCl/NaCl solution was removed from each container.

[0043] C. To each container was then added approximately 1 L solution of0.22 μm filter sterilized 1 M sodium chloride (NaCl)/10 mM phosphatebuffered saline (PBS). Containers were then placed on a shaker table forapproximately 18 hours at 200 rpm. After shaking, the NaCl/PBS solutionwas removed from each container.

[0044] D. To each container was then added approximately 1 L solution of0.22 μm filter sterilized 10 mM PBS. Containers were then placed on ashaker table for about two hours at 200 rpm. After shaking, thephosphate buffered saline was then removed from each container.

[0045] E. Finally, to each container was then added approximately 1 L of0.22 μm filter sterilized water. Containers were then placed on a shakertable for about one hour at 200 rpm. After shaking, the water was thenremoved from each container.

[0046] Processed ICL samples were cut and fixed for histologicalanalyses. Hemotoxylin and eosin (H&E) and Masson trichrome staining wasperformed on both cross-section and long-section samples of both controland treated tissues. Processed ICL samples appeared free of cells andcellular debris while untreated control samples appeared normally andexpectedly very cellular.

Example 2

[0047] Comparative Study of Other Cleaning Treatments for CollagenousTissue

[0048] Other methods for disinfecting and sterilizing collagenoustissues described in U.S. Pat. No. 5,460,962 to Kemp were compared tosimilar methods described by Cook. et al. in International PCTapplication WO 98/22158. Examples 1, 2, and 3, from Kemp, in addition toa non-buffered peracetic acid method were done.

[0049] Small intestines were harvested from 4 large pigs. Intestineswere procured, the outer mesenteric layer was stripped, and theintestines were flushed with water.

[0050] The study included seven conditions: Condition A was carried outaccording to the disclosure of Example 1 in Cook, et al. inInternational PCT Application WO 98/22158. Condition B was a variationof A in that the intestinal material was mechanically cleaned beforeemploying the disclosed chemical treatment. Conditions C, D, and E werecarried out according to the methods of Examples 1, 2, and 3 in U.S.Pat. No. 5,460,962 to Kemp. In all conditions, a ten-to-one ratio ofsolution to material is used, that is, 100 g of tissue material istreated with 1 L of solution.

[0051] A. Material from each of the 4 intestines were placed intoseparate bottles (n=5) containing a one liter solution of 0.2% peraceticacid in 5% ethanol (pH 2.56) and agitated on a shaker platform. Aftertwo hours of agitation, condition A was mechanically cleaned on theBitterling gut cleaning machine.

[0052] For the other six conditions, B through G, intestine wasmechanically cleaned using the Bitterling gut cleaning machine prior tochemical treatment. After mechanical cleaning, representative piecesfrom the 4 intestines were placed into bottles containing solution forchemical treatment. Bottles were shaken 18±2 hours on a platform. Theremaining six conditions, B through G, were as follows:

[0053] B. A one liter solution of 0.2% peracetic acid in 5% ethanol (pH2.56) (n=5).

[0054] C. A one liter solution of 0.1% peracetic acid in phosphatebuffered saline (pH 7.2) (n=3).

[0055] D. A one liter solution of 0.1% peracetic acid and 1 M sodiumchloride (NaCl) (pH 7.2) (n=3).

[0056] E. A one liter solution of 0.1% peracetic acid and 1 M NaCl (pH2.9) (n=3).

[0057] F. One liter solution of “chemical cleaning” solutions asmentioned above in Example I (n=4).

[0058] G. A one liter solution of 0.1% peracetic acid in deionizedwater, buffered to pH 7.0 (n=2).

[0059] After chemical and mechanical treatments, all conditions wererinsed for a total of 4 times with filtered sterile purified water. Themechanically and chemically treated material was grossly stained toexamine cellular debris with Mayer's hematoxylin. Morphologicalassessment included Hematoxylin & Eosin, Masson's Trichrome, andAlizarin Red staining techniques. Histological results from the varioustreatments show that the method of condition A yielded a material whereit was difficult to remove mucosal layers on Bitterling after chemicaltreatment. The material had to be run through Bitterling about an extra10-12 times. The material was very swollen at first and had asignificantly large amount of cellular debris on surface and in thevasculature of the material. The method of condition B was also veryswollen and also demonstrated a significantly large amount of cellulardebris on surface and in the vasculature of the material. The methods ofconditions C and D yielded a non-swollen material having minimalcellular debris in vasculature. Condition E yielded a material that wasslightly swollen and contained minimal cellular debris in thevasculature.

[0060] A DNA/RNA isolation kit (Amersham Life Sciences) was used toquantify the residual DNA/RNA contained in the cleaned tissues. Theresults are summarized in Table 1. TABLE 1 DNA/RNA Isolation kit Results(μg DNA/mg tissue) Condition A B C D E F G Average ± 2.16 ± 0.32 2.1 ±0.48 0.32 ± 0.11 1.92 ± 0.28 0.32 ± 0.23 0 ± 0 1.42 ± 0.03 Std. Dev.

[0061] Morphological analysis correlates with the DNA/RNA quantificationto show that the cleaning regimens of conditions A and B result in acollagenous tissue matrix that remains highly cellular and containresidual DNA as a result. The cleaning methods of Kemp are much moreeffective for the removal of cells and cellular debris from collagenoustissue matrices. Finally, the chemical cleaning method of Condition F,described in International PCT Application No. WO 98/49969 to Abraham,et al. and outlined in Example 1, above, removes all cells and cellulardebris and their DNA/RNA to a level undetectable by these methods.

Example 3

[0062] Method for Making an ICL Tube Construct

[0063] In the sterile field of a laminar flow cabinet, the ICL wasformed into ICL collagen tubes by the following process. Lymphatic tagswere trimmed from the serosal surface of the ICL. The ICL was blottedwith sterile absorbent towelettes to absorb excess water from thematerial and then spread on a porous polycarbonate sheet and dried inthe oncoming airflow of the laminar flow cabinet. Once dry, ICL was cutinto 28.5 mm×10 cm pieces for a 2 layer graft with approximately a 10%overlap. To support the ICL in the formation of the tubes, a cylindricalstainless steel mandrel with a diameter of about 4 mm was covered withKRATON®, an elastic sleeve material that facilitates the removal of theformed collagen tube from the mandrel and does not adhere or react withthe ICL. The long edge of the ICL was then moistened with sterile waterand adhered to the mandrel and allowed to dry for about 15 minutes toform a “flag”. Once adhered, the ICL was rolled around the mandrel andover itself one complete revolution. After rolling was complete, airbubbles, folds, and creases were smoothed out from under the materialand between the layers. The mandrels and rolled constructs were allowedto dry in the oncoming airflow of the laminar flow cabinet for about anhour in the cabinet at room temperature, approximately 20° C.

[0064] Chemical crosslinking solution of either crosslinked 1 mM EDC or10 mM EDC/25% acetone v/v in water, in volumes of about 50 mLcrosslinking solution per tube, was prepared immediately beforecrosslinking; EDC will lose its activity over time. The hydrated ICLtubes were then transferred to either of two cylindrical vesselscontaining either crosslinking agent. The vessel was covered and allowedto sit for about 18±2 hours in a fume hood, after which time thecrosslinking solution was decanted and disposed. ICL tubes were thenrinsed three times with sterile water for about 5 minutes per rinse.

[0065] The crosslinked ICL tubes were then removed from the mandrel bypulling the Kraton sleeve off the mandrel from one end. Once removed,the ICL tube containing the Kraton were allowed to dry for an hour inthe hood. Once dried, the sleeve was removed from the lumen of the ICLtube simply by pulling it out from one end.

[0066] ICL tubes were sterilized in 0.1% peracetic acid at approximatelypH 7.0 overnight according to the methods described in commonly ownedU.S. Pat. No. 5,460,962, the disclosure of which is incorporated hereinin its entirety. The ICL tubes were then rinsed of sterilizationsolution three times with sterile water for about 5 minutes per rinse.The peracetic acid sterilized ICL collagen tubes were then dried in thelaminar flow hood and then packaged in sterile 15 mL conical tubes untilimplantation.

Example 4

[0067] Mechanical testing of ICL Tube Prostheses

[0068] Various mechanical properties of a 2 layer ICL tubular constructformed from a single sheet of ICL wrapped around a mandrel with 20%overlap, crosslinked at lmM EDC in water was measured. Suture retention,burst. porosity (leakage/integral water permeability), and compliancetesting were done in accordance with the “Guidance for the Preparationof Research and Marketing Applications for Vascular Graft Prostheses”,FDA Draft Document. August 1993. Suture retention, burst and complianceanalyses were performed using a servohydraullic MTS testing system withTestStar-SX software. Results are summarized in Table 2.

[0069] Briefly, the suture retention test consisted of a suture beingpulled 2.0 mm from the edge of a graft at a constant rate. The peakforce when the suture ripped through the graft was measured. The averagemeasurement obtained was above required limits indicating that theconstruct can withstand the physical pressures of suturing in theclinic.

[0070] In the burst test, pressure was applied to the graft in 2.0 psiincrements for one minute intervals until the graft burst. Forreference, systolic pressure is approximately 120 mmHg (16.0 kPa) in anormotensive person, thus the burst strength obtained by the testingdemonstrated that the construct could maintain pressures about 7.75times systolic pressure thus indicating that the construct can begrafted for vascular indications and withstand the rigors bloodcirculation.

[0071] For compliance testing, the graft was brought to 80 and 120 mmHgin succession. The diameter of the graft was then measured at eachpressure using image analysis software and the compliance calculated as(D₁₂₀−D₈₀)/(D₈₀×40mmHg)×100%. Compliance of a rabbit carotid artery isapproximately 0.07% /mmHg, human artery is about 0.06% /mmHg and humanvein is about 0.02% /mmHg, indicating that the construct exhibits therequisite compliance to serve as a vascular graft.

[0072] To measure porosity, PBS under hydrostatic pressure of 120 mmHgis applied to the graft. The volume of PBS that permeated through thegraft over a 72 hour period was normalized to the time and surface areaof the graft to calculate the porosity.

[0073] The shrink temperature is used to monitor the extent ofcrosslinking in a collagenous material. The more crosslinked a graft,the more energy is required, thus a higher shrink temperature. Adifferential scanning calorimeter was used to measure the heat flow toand from a sample under thermally controlled conditions. The shrinktemperature was defined as the onset temperature of the denaturationpeak in the temperature-energy plot.

[0074] The suture retention is well above the 2 N suggested for suturinga prosthesis in a patient; a surgeon's pull force when suturing is about1.8 N. The burst strength over seven times systolic pressure. Thecompliance is in the range of human arteries and veins. The porosity ofthe ICL tube is low compared to a woven graft: the ICL tube does notrequire pre-clotting. The shrink temperature, a measure of the collagendenaturation temperature, is close to that of non cross-linked ICLindicating a low amount of cross-linking. Mechanical testing wasperformed on the ICL sleeve prosthesis to determine the strength of theICL sleeve. A summary of results from the various tests of mechanicaland physical characteristics of 2-layer ICL constructs are presented inTable 2. TABLE 2 Summary of Mechanical Properties Mechanical Test ResultSuture Retention Test 3.97 ± 0.7 N Burst Test 18.0 ± 5.4 psi(124 ± 37kPa) Porosity  3.4 × 10⁻⁴ ml/cm²min Shrink Temperature 68.4 ± 0.4° C.Compliance (between 80 and 120 mmHg) 0.05 %/mmHg

Example 5

[0075] Implantation of Collagen Tubes as External Stents

[0076] Twenty-nine New Zealand male white rabbits underwentinterposition bypass grafting of the right common carotid artery usingthe reversed ipsilateral jugular vein. In the experimental group (n=15),once the proximal anastomosis was performed, the vein was passed througha collagen tube having dimensions of 4 mm in diameter and 35 to 40 mm inlength and the distal anastomosis was then completed. Leaks wererepaired and the collagen tube was fashioned to completely cover thevein graft, including both anastomoses. Control animals (n=14) weretreated identically but without tube support. One intraoperative deathresulted from an unrecognized leak in the mid-segment of a vein graft inthe experimental group. Otherwise, there were no other significantcomplications such as infection or bleeding in either group. All animalssurvived until end-points and all vein grafts were patent at harvest.Postoperatively, the flow rate and intraluminal pressure in vein graftswere measured on either day 3 or 28 (n=5 per group). Vein grafts wereharvested on day 3 for assessment of tyrosine phosphorylation by Westernblot analysis (n=4 per group), and on day 28 for morphometricmeasurement (n=5 per group), scanning and transmission electronmicroscopy (n=5 per group) and isometric tension studies (n=5 pergroup). On the day of harvest, animals were anesthetized andsubsequently sacrificed with an intravenous overdose of barbiturates.

[0077] Vein grafts implanted in the arterial circulation predictablydevelop wall thickening, with smooth muscle cell hyperplasia anddeposition of extracellular matrix in the intima and media, an adaptiveprocess that has been referred to as “arterialization”. In 50% ofimplanted vein grafts, however, this process becomes pathologic usuallydue to intimal hyperplastic lesions causing either focal stenosis orpromoting accelerated atherosclerosis. This study shows that externaltube support of vein grafts effectively modulates tyrosine kinasesignaling and the hyperplastic response in experimental vein grafts,with increased shear stress and reduced wall tension.

Example 6

[0078] Hemodynamic Assessment

[0079] The rate of blood flow was measured by applying flow probes (3 or4 mm diameter), connected to flowmeter (Transonic Systems Inc., Ithaca,N.Y.), onto the external surface of the vessels; flow was measured withthe collagen tube in situ in tube-supported vein grafts. Theintraluminal blood pressure was measured using a 27-gauge needle,connected to a pressure transducer and monitor (Propaq 106, ProtocolSystems Inc., Beaverton, Oreg.). Flow rates and intraluminal pressureswere determined in the carotid artery (proximal and distal to the veingraft) and in the vein graft in a pilot study; there was no significantdifferences in the flow rates or pressure levels in vein grafts comparedto the proximal or distal segments of carotid arteries. Hence, valuesreported for flow rate (Q; in ml·min⁻¹) were taken from the mid-segmentsof vein grafts and values for intraluminal blood pressure (P; in mmHg)from the proximal segments of the carotid arteries.

[0080] Shear stress was calculated as τ=4ηQ/πr_(i) ³ in dyne/cm² (τ,shear stress; η, blood viscosity; Q, flow rate; r_(i), internal radius).Wall tension was calculated as T=P·r_(i) in 10³ dyne/cm¹ (T, walltension; P, mean arterial blood pressure; r_(i), internal radius). Theblood viscosity (0.03 in poise) was assumed to be constant. The internalradius (r_(i)) was determined by morphometry; we previously demonstratedthat histologic diameter underestimated the in situ diameter by 10%. Foranalytical purposes, the internal radii and wall tensions wererecognized as approximations and the flow of blood was assumed to belaminar. To normalize the wall tension by wall thickness, the walltensile stress was also calculated (wall tensilestress=pressure×internal radius/wall thickness). Wall thickness wasdefined as the sum of the thickness of the intima, the media, and thecollagen tube, respectively.

[0081] Flow rates and pressures were not significantly altered in veingrafts with tube support as compared to controls (Table 3). Applying theequations formulated in above, the calculated wall tension was decreasedby 1.7-fold and shear stress was increased by 4.8-fold in tube supportedvein grafts compared to controls (Table 3). The decrease in wall tensionwas expected because the pressure was not different but the internalradius was reduced by 1.7-fold in tube supported vein grafts compared tocontrols (1.63±0.06 mm vs. 2.69±0.09mm, respectively; p<0.0001).Similarly, the increase in shear stress was anticipated since flow wasnot significantly changed and shear stress is inversely proportional tothe third power of the internal radius.

[0082] Hemodynamic forces are known to play an important role in theregulation of cells that compose the blood vessel wall. In particular,the effects of shear stress on endothelial cells have been studiedextensively in vitro. Several shear stress-inducible endothelial geneshave been identified in vitro, including PDGF-A, PDGF-B, basicfibroblast growth factor (FGF) and nitric oxide synthase, all of whichhave been implicated in wound remodeling. The transformation ofbiomechanical (hemodynamic) stimuli into biological responses usuallybegins with the activation of protein kinases and protein-to-proteininteractions leading to gene transcription (or inhibition thereof).Takahashi and Berk, J Clin Invest. 34: 212-219 (1996), have demonstratedthat shear stress can activate extracellular signal-regulated kinase(ERK1/2) via a tyrosine kinase-dependent pathway in cultured humanumbilical vein endothelial cells. The hemodynamic factors in vivo arecomplex, however, the relative importance of each of these factors hasbeen identified in animal models. TABLE 3 Hemodynamic Parameters. TubeSupport Control p-value Flow (ml · min⁻¹) 12.8 ± 1.1 11.5 ± 1.0 0.41Pressure (mmHg) 53.2 ± 3.8 57.8 ± 1.4 0.29 Wall Tension (×10³ 11.7 ± 0.919.8 ± 0.5 <0.01 dyne · cm⁻¹) Shear Stress  1.9 ± 0.25  0.4 ± 0.04<0.001 (dyne · cm⁻²)

Example 7

[0083] Protein Extraction and Western Blot Analysis

[0084] Excised vein grafts were cleared of adventitial tissues, washedin ice cold phosphate buffered saline (PBS), cut into 1 cm rings, snapfrozen in liquid nitrogen and stored at −80 C. Proteins were extractedfrom the frozen samples by grinding the tissues to a fine powder in amortar and pestle in liquid nitrogen followed by sonication in ice-coldlysis buffer (1:4 w:v; 50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodiumdeoxycholate, 150 mM NaCl, 1 mM EGTA, 1 mM PMSF, 1 mM sodiumorthovanadate, 1 mM sodium fluoride, 1 μg·ml⁻¹ aprotinin, 1 μg·ml⁻¹leupeptin, and 1 μg·ml⁻¹ pepstatin). Insoluble debris was pelleted in amicrocentrifuge at 14,000 g at 4° C. The supernatant was collected ascell lysates and stored at −80 ° C. until used. Protein concentrationwas determined using Bradford assay (Biorad Laboratories, Richmond,Calif.) with bovine serum albumin (BSA) as the standard.

[0085] Equal amounts of protein extracts (15 μg) were mixed in a gelloading buffer (20% glycerol, 100 mM Tris-HCl pH 7.4, 100 mM NaCl, 100mM dithiothreitol) (1:4; v/v) and boiled for 9 minutes. Samples werethen loaded onto an 8% SDS-polyacrylamide minigel, separated byelectrophoresis and transferred onto a nitrocellulose membrane.Non-specific binding was blocked by incubating the membrane in TTBS (10mM Tris-HCl, pH 8.0, 0.05% TWEEN-20 and 150 mM NaCl) containing 1% BSAovernight at 4° C. A monoclonal mouse anti-phosphotyrosine antibody(PY20, 1 μg·ml; Chemicon International Inc., Temecula, Calif.) was thenapplied to the blot for 1 hour at room temperature. Antibody binding wasdetected by incubating the blot with a horseradish peroxidase conjugatedgoat anti-mouse IgG (1:5000 dilution; Santa Cruz Biotechnology, SantaCruz, Calif.). The blot was washed several times between blocking stepswith TTBS. The immunoblot was visualized using an enhancedchemiluminescence kit (Amersham, Arlington Heights, Ill.) andautoradiographed. The autoradiographs were scanned, analyzed (AdobePhotoshop 3.0, Adobe Systems Inc., Mountain View, Calif.) and theintegrated density of visualized bands was measured (N.I.H Image 1.61).Chemicals were obtained from Sigma Chemical Co. (St Louis, Mo.) unlessotherwise stated.

[0086] Western blot analysis demonstrated a 15-fold reduction (p<0.001)in phosphorylated tyrosine residues in the wall extracts of day 3 tubesupported vein grafts, when compared to controls. Phosphorylatedtyrosine residues were detected in approximately 113 kDa proteins intube supported vein grafts. In control vein grafts, however, in additionto the greater amount of phosphorylated tyrosine residues inapproximately 113 kDa proteins, phosphorylated tyrosine residues werealso present in proteins with molecular weights just above 82 kDa and of200 kDa.

[0087] Protein tyrosine kinase activity is markedly reduced in veingrafts with reduced wall tension and increased shear stress, both ofwhich are the consequences of the tube support. The identity of thetyrosine phosphorylated proteins (of approximately 82, 113 and 200 kDa)remains to be further defined. However, we postulate that the decreasedtyrosine kinase activity in tube supported vein grafts may, in part, beassociated with reduced expression or activation of the receptors forgrowth factors, such as PDGF, FGF and epidermal growth factor; thereceptors for these growth factors have intrinsic protein tyrosinekinases, which range from 110 to 170 kDa in molecular weight. Moreover,Kraiss et al. (Circ Res 1996;79:45-53) have shown that abrupt reductionin both blood flow and shear stress is associated with increased PDGF-AmRNA and protein expression in baboon prosthetic grafts. In parallel,Mehta et al. (Nature Medicine 1998;4:235-239) have recently demonstrateda significant decrease in PDGF-B protein with external stenting of veingrafts in the pig model. Although wall tension and shear stress were notassessed in the study of Mehta et al, supra, their external stent modellikely produced hemodynamic effects similar to our tube support model,that is, reduced wall tension and increased shear stress.

Example 8

[0088] Morphologic Assessment

[0089] Vein grafts were cleared of blood with an initial infusion ofHanks Balanced Salt Solution (Gibco Laboratories, Life TechnologiesInc., Grand Island, N.Y.). As previously described, vein grafts werethen perfused fixed in situ with 2% glutaraldehyde made up in 0.1Mcacodylate buffer (pH 7.2) supplemented with 0.1M sucrose to give anosmolality of approximately 300 mOsm, at a pressure of 80 mmHg. Afterimmersion in the fixative for 48 hours, cross-sections (3 per graft)from the middle segment of the vein grafts were processed formorphometric assessment. Briefly, morphometric assessment was performedon sections that were stained with a modified Masson's trichrome andVerhoeff's elastin stain. The intima and media were delineated byidentification of the demarcation between the criss-cross orientation ofthe intimal hyperplastic smooth muscle cells and circular smooth musclecells of the media. The outer limit of the media was defined by theinterface between the circular smooth muscle cells of the media and theconnective tissue of the adventitia. The dimensions of the lumen, intimaand media were measured by videomorphometry (Innovision 150, AmericanInnovision Inc., San Diego, Calif.). The internal radius and thethickness of the intima and media of vein grafts were derived from themeasured luminal intimal and medial areas. The intimal ratio (intimalratio=intimal area/[intimal+medial areas]) and luminal index (luminalindex=luminal diameter/[intimal+medial thicknesses]) were alsocalculated.

[0090] After further specimen processing as previously described,scanning electron microscopy (Philips 500 scanning electron microscope,N. V. Philips, Eindhoven, The Netherlands) and transmission electronmicroscopy (Philips 300 transmission electron microscope, N. V. Philips,Eindhoven, The Netherlands) were performed on representativemid-sections.

[0091] Externally supporting vein grafts with the collagen tube reducedthe luminal diameter of day 28 vein grafts by 63% compared to controlvein grafts (Table 4). The thickness of the intima was decreased by 45%(46±2 μm vs 84+5 μm, p<0.0001) and the media by 20% (63±8 μm vs 79±4 μm,p<0.05) in tube supported vein grafts compared to controls,respectively. Both intimal and medial areas were also reduced, 66% and49%, respectively (Table 4). Due the greater reduction in intimaldimension relative to the reduction in the media, the intimal ratio wasdecreased by 10% (Table 4). However, the luminal index, an assessment ofcross-sectional wall thickness relative to luminal diameter, wasmaintained constant with or without tube support (Table 4).

[0092] Scanning electron microscopy showed a confluent endotheliallining with distinct cell borders in both tube supported vein grafts andcontrol vein grafts. Endothelial cells were unaltered and flattened intube supported vein grafts compared to more cuboidal and bulgingendothelial cells in the control vein graft. On transmission electronmicroscopy, vein grafts with tube support had less subendothelial edemaand less debris than controls; additionally, the orientation of intimalsmooth muscle cells were orderly and circular, and their shape waselongated and organized in several layers in tube supported vein grafts.In contrast, intimal smooth muscle cells were disorganized and lesselongated in control vein grafts.

[0093] A multitude of hemodynamic factors are known to influence wallthickening in vein grafts. Schwartz, et al (J Vasc Surg 1992;15:176-186)have shown that “myointimal” (referring to both intima and media)thickening correlates most strongly with wall tension in rabbit veingrafts. On the other hand, Dobrin (Hypertension 1995;26:38-43) hasdemonstrated that intimal thickening correlates best with low flowvelocity (a determinant of shear stress) and that medial thickening wasa better correlate of deformation in the circumferential direction (adeterminant of wall tension). The prevailing concept is that wallremodeling is dependent on both shear stress and wall tension. In thisstudy, we found a greater reduction in intimal thickening than medialthickening which may correlate with the larger increase in shear stressand smaller decrease in wall tension, respectively, which would supportDobrin's results. Although wall thickening is due to both hyperplasia ofsmooth muscle cells and elaboration of an extracellular matrix, more isknown about the former than latter. Zwolak, et al. (J Vasc Surg1987;5:126-136) have described the cellular kinetics in the rabbit veingrafts. The proliferation of smooth muscle cells has been shown toincrease in grafts subjected to low flow and shear stress. Additionally,Mehta, et al, supra, have reported that stenting of vein grafts reducesintimal and medial smooth muscle cell proliferation as assessed byimmunostaining for the proliferating cell nuclear antigen (PCNA). TABLE4 Dimensional Analysis of Day 28 Vein Grafts. Tube Support Controlp-value Luminal area (mm²)  8.6 ± 0.6 23.2 ± 1.6 <0.001 Intimal area(mm²) 0.48 ± 0.02 1.42 ± 0.08 <0.001 Medial area (mm²) 0.70 ± 0.11 1.36± 0.07 <0.001 Intimal ratio 0.46 ± 0.06 0.51 ± 0.01 <0.01 Lumnial index34.1 ± 3.6 34.9 ± 22 0.33

[0094] Statistical differences between tube supported vein grafts andcontrol vein grafts were compared using unpaired Mann-Whitney Rank sumtest.

Example 9

[0095] Isometric Tension Studies

[0096] Vein grafts were sectioned into four 5 mm rings. In the tubesupported group, the collagen tube was carefully dissected off andremoved to allow unimpeded vessel contraction and relaxation. Each ringwas immediately mounted between two stainless steel hooks in 5 ml organbaths containing oxygenated Krebs solution (122 mM NaCl, 4.7 mM KCl, 1 2mM MgCl₂, 2.5 mM CaCl₂, 15.4 mM NaHCO₃, 1.2 mM KH₂PO₄ and 5.5 mMglucose, maintained at 37° C. and oxygenated with 95% O₂ and 5% CO₂), aspreviously described with some modifications. In brief, followingequilibration, the resting tension was adjusted in increments from 0.5to 1.25 gms and the maximal response to a modified oxygenated Krebssolution containing 60 mM KCl, 66.7 mM NaCl, 1.2 mM MgCl₂, 2.5 mM CaCl₂,15.4 mM NaHCO₃, 1.2 mM KH₂PO₄ and 5.5 mM glucose was measured toestablish a length-tension relationship. Cumulative dose response curvesto the contractile agonists bradykinin (10⁻⁹ to 10⁻⁵ M), norepinephrine(10⁻⁹ to 10⁻⁴ M), and serotonin (10⁻⁹ to 10⁻⁴ M) were performed.Relaxation responses to acetylcholine (10⁻⁸ to 10⁻⁴ M), an endotheliumdependent agonist, and nitroprusside (10⁻⁸ to 10⁻⁴ M), an endotheliumindependent agonist, were assessed on rings precontracted withnorepinephrine, at the concentration which produced 80% of maximalcontraction. All rings were allowed to re-equilibrate for a minimum of30 minutes between each experimental run and the same sequence ofagonist testing was maintained for all experiments. (All chemicals wereobtained from Sigma Chemical Co. (St Louis, Mo.)).

[0097] Tube supported vein grafts demonstrated similar responses to KClcompared to controls (force: 300±46 mg vs 280±47 mg). The sensitivitiesof tube supported vein grafts in response to norepinehrine and serotoninwere not significantly different than that of controls (Table 5). Tubesupported vein grafts were, however, more sensitive to bradykinin thancontrols (Table 5). The maximal contractile forces generated in responseto all three agonists (norepinephrine, serotonin and bradykinin),expressed as standardized contractile ratios, were not significantlyaltered with external tube support of vein grafts.

[0098] As previously reported, control vein grafts did not relax inresponse to acetylcholine. In contrast, 10 of 20 rings from tubesupported vein grafts demonstrated dose-dependent relaxation in responseto acetylcholine with a maximal relaxation to 64% of precontractedtension, albeit with a low sensitivity (Table 5). Of the five tubesupported vein grafts studied, only one had no response to acetylcholinein all rings. In response to nitroprusside, the sensitivity (Table 5)and maximal relaxation were similar in vein grafts with or without tubesupport.

[0099] These results show complete preservation of smooth muscle cellfunction and recovery of endothelial-dependent relaxation with tubesupport of vein grafts. Despite the significant reduction in wallthickness, tube supported vein grafts generated similar contractileforces in response to KCl and all three contractile agonists tested(norepinephrine, serotonin and bradykinin). The maximal force generatedby a vessel ring can be correlated with smooth muscle cell mass,provided that all other factors (such as the integrity and number ofreceptors for the agonist or potassium channels) are constant. It wouldfollow that smooth muscle cell mass was not significantly changed withtube support, suggesting that the reduction in intimal thickness may inpart be due to decreased production of extracellular matrix. TABLE 5Vasomotor Responses of Day 28 Vein Grafts. Tube Support Control p-valueNorepinephrine 5.96 ± 0.07 5.97 ± 0.06 0.91 Serotonin 6.39 ± 0.11 6.28 ±0.07 0.22 Bradykinin 6.32 ± 0.08 5.60 ± 0.09 <0.001 Acetylcholine 3.92 ±0.22 no response <0.01 Nitroprusside 6.46 ± 0.12 6.73 ± 0.19 0.25

[0100] The concentration for the half maximal response (EC₅₀) wascsalculated by logistic analysis and the sensitivity is defined as−log₁₀(EC₅₀). In each vein graft, the sensitivity was determined foreach vessel ring (4 rings per vein graft) and the mean was taken as thevalue for that vein graft. Values shown are the mean±s.e.m (n=5 pergroup). Statistical differences between the tube supported vein graftsand control vein grafts were compared using the unpaired Student'st-test.

[0101] The recovery of endothelium-dependent relaxation to acetylcholinein 50% of vessel rings with tube support would indicate that endothelialfunction was modulated. Increased shear stress has been shown tostimulate increased production of nitric oxide in vitro, which mayexplain in part the relaxation to acetylcholine in tube supported veingrafts. Systemic supplementation with L-arginine, the nitric oxideprecursor, has also been shown to preserve endothelial-dependentrelaxation of vein grafts to acetylcholine. Improved endothelialfunction has also been reported by Onohara, et al (J Surg Res1993;55:344-350) with increased prostacyclin (PGI) production in veingrafts exposed to high shear stress. Alternatively, the preservedendothelial function in vein grafts may be attributed to the lesser wallstretch injury with tube support. All in all, endothelial cells areknown to have regulatory role in smooth muscle cell proliferation andmigration in addition to its role in mechanotransduction and vasomotorresponses. We therefore postulate that improved endothelial functionwith tube support may reduce the release of mitogenic andchemoattractant signals such as PDGF.

[0102] Although the foregoing invention has been described in somedetail by way of illustration and example for purposes of clarity andunderstanding, it will be obvious to one of skill in the art thatcertain changes and modifications may be practiced within the scope ofthe appended claims.

We claim:
 1. A bioremodelable prosthesis comprising a collagen tubecomprising at least one layer of processed tissue matrix of crosslinkedsubmucosal collagen of small intestine.
 2. The bioremodelable prosthesisof claim 1, wherein the prosthesis functions as a remodeling templatefor the ingrowth of host cells.
 3. The bioremodelable prosthesis ofclaim 1, wherein the processed tissue matrix is bonded to itself oranother layer of processed tissue matrix.
 4. The bioremodelableprosthesis of claim 1, wherein the processed tissue matrix is derivedfrom the tunica submucosa of mammalian small intestine.
 5. Thebioremodelable prosthesis of claim 4, wherein the processed tissuematrix is derived from the tunica submucosa of porcine small intestine.6. The bioremodelable prosthesis of claim 1, wherein the collagen tubecomprises at least two layers of processed tissue matrix of crosslinkedsubmucosal collagen.
 7. The bioremodelable prosthesis of claim 6,wherein the layers are derived from the same material.
 8. Thebioremodelable prosthesis of claim 6, wherein the layers are derivedfrom the different collagen materials.
 9. The bioremodelable prosthesisof claim 6, wherein the layers are crosslinked.
 10. The bioremodelableprosthesis of claim 1, wherein the collagen tube defines a lumen havinga luminal surface, wherein the processed tissue matrix has a mucosalsurface and a serosal surface, and wherein the mucosal surface of theinnermost layer of processed tissue matrix is the luminal surface of thecollagen tube.
 11. The bioremodelable prosthesis of claim 1, wherein thebioremodelable prosthesis is an external vein support.
 12. Thebioremodelable prosthesis of claim 1, wherein the bioremodelableprosthesis is a neuron growth tube.
 13. A bioremodelable prosthesiscomprising a collagen tube comprising at least one layer of processedtissue matrix of crosslinked submucosal collagen of small intestine,wherein the prosthesis functions as a remodeling template for theingrowth of host cells, wherein the prosthesis is pliable, suturable,non-creeping, semi-permeable, and non-porous, and wherein the prosthesisis sterilized.
 14. A bioremodelable external vein support comprising acollagen tube comprising at least one layer of processed tissue matrixof crosslinked submucosal collagen of small intestine.
 15. Thebioremodelable external vein support of claim 14, wherein collagen tubedefines a lumen having a luminal surface, wherein the processed tissuematrix has a mucosal surface and a serosal surface, and wherein themucosal surface of the innermost layer of processed tissue matrix is theluminal surface of the collagen tube.
 16. A neuron growth tubecomprising a collagen tube comprising at least one layer of processedtissue matrix of crosslinked submucosal collagen of small intestine. 17.The neuron growth tube of claim 16, wherein the neuron growth tubefunctions to guide nerve regeneration.
 18. The neuron growth tube ofclaim 16, wherein the neuron growth tube contains extracellular matrixcomponents, at least one growth factor, cultured cells, or a combinationthereof.